Berliner und Münchener Tierärztliche Wochenschrift 2020

Chemisches und Veterinäruntersuchungsamt Stuttgart (CVUAS), Fellbach1 Open Access Konsiliarlabor für pseudotuberculosis (DVG), Fellbach2 Wilhelma – Zoologisch-Botanischer Garten, Stuttgart3 Berl Münch Tierärztl Wochenschr DOI 10.2376/1439-0299-2020-6 Actinomycetes associated with for- © 2020 Schlütersche mation in a goat, a llama and two alpacas Verlagsgesellschaft mbH & Co. KG ISSN 1439-0299 Actinomyceten im Zusammenhang mit Abszessen bei Korrespondenzadresse: einer Ziege, einem Lama und zwei Alpakas [email protected]

Eingegangen: 19.02.2020 Reinhard Sting1,2, Anna Katharina Schwalm1, Matthias Contzen1, Marco Roller3, Angenommen: 15.05.2020 Jörg Rau1 Veröffentlicht: 16.07.2020 https://www.vetline.de/berliner-und- muenchener-tieraerztliche-wochenschrift- open-access

Abstract The genera and Schaalia, both members of the bacterial order Actino- mycetales, include relevant infectious agents that cause in small rumi- nants and New World camelids. Due to the high diversity of the , detection of undescribed members of this order is to be expected. Novel actinomy- cetes species were cultivated from a goat, a llama and two alpacas suffering from abscesses with suspected caseous lymphadenitis (CLA). Analyses carried out on these isolates using MALDI-TOF MS and 16S rRNA gene sequencing revealed actino- mycetes, presumably belonging to the bacterial genera Actinomyces and Schaalia. The data suggest that the caprine isolate is a undescribed Actinomyces species, while the isolates originating from a llama and two alpacas show a close relation- ship to each other within a unique Schaalia cluster, suggesting a host-adapted novel Schaalia species. Both methods proved equally suitable for reliable identification of known and of undescribed Actinomyces and Schaalia species. This study contributes to extending our knowledge about novel species belonging to the bacterial family of (actinomycetes) associated with abscesses in goats and New World camelids. Precise identification of actinomycetes at species level is of high relevance in veterinary practice with regard to differentiation from caseous lymphad- enitis and assessment of treatment success.

Keywords: small ruminant, New World camelids, pseudotuberculosis, Actinomyces, Actinomycetes, Actinomycetaceae, Schaalia

Zusammenfassung Bakterien der Gattungen Actinomyces und Schaalia gehören zur Ordnung Actino- mycetales und sind wichtige Erreger infektiöser Abszesse bei Wiederkäuern und Neuweltkameliden. Aufgrund des großen Artenreichtums der Actinomycetales sind neue und bisher nicht beschriebene Arten zu erwarten. So konnten wir neue Acti- nomyceten aus Abszessen einer Ziege, eines Lamas und zweier Alpakas mit Verdacht auf Pseudotuberkulose isolieren. Untersuchungen der Isolate mittels MALDI-TOF- Massenspektrometrie und Sequenzierungen des 16S rRNA-Gens ergaben Actino- myceten, die den Gattungen Actinomyces und Schaalia zugeordnet werden können. Die Ergebnisse weisen darauf hin, dass es sich bei dem Ziegen-Isolat um eine bisher noch nicht beschriebene Actinomyces-Spezies handelt. Die Isolate von dem Lama und den beiden Alpakas hingegen erwiesen sich als eng verwandt innerhalb eines gemeinsamen Schaalia-Clusters, was auf eine neue wirtsadaptierte Schaalia-Spezies hinweist. Beide Methoden erwiesen sich als geeignet, bekannte und bisher nicht beschriebene Actinomyces- und Schaalia-Spezies zuverlässig zu identifizieren. Diese Studie trägt dazu bei, unsere Kenntnisse über neue Spezies der Familie der Actinomycetaceae (Actinomyceten) im Zusammenhang mit Abszessen bei Ziegen und Neuweltkameliden zu erweitern. Eine exakte Identifizierung von Actinomyce- ten ist unter dem Hintergrund der Differenzierung zur Pseudotuberkulose und der Einschätzung von Therapieerfolgen von großer Bedeutung.

Schlüsselwörter: Kleine Wiederkäuer, Neuweltkameliden, Pseudotuberkulose, Actinomyces, Actinomyzeten, Actinomycetaceae, Schaalia Berliner und Münchener Tierärztliche Wochenschrift 2020

Introduction In October 2018, an adult goat (Caprae aegagrus hircus) suffered from a neck abscess. Material for bacteriological Abscess formation due to bacterial infections can lead examination was taken from the opened abscess. to the development of severe debilitating or even life- In August 2019, an abscess was noticed on the shoul- threatening diseases in animals and humans. Among der and chest area of an alpaca (Vicugna pacos). After causative pathogens, of the order Actinomy- opening the abscess, material was taken for bacteriolog- cetales are of special relevance for purulent lesions. ical examination. Actinomycetales belong to the , which In September 2019, another adult alpaca had three represent a diverse phylum of gram-positive bacteria, abscesses, two in the left cheek and one at the left jaw comprising the major pathogen containing bacterial angle. The abscesses were opened by incision and a families Actinomycetaceae, Corynebacteriaceae, Mycobac- smooth, yellowish mass was recovered and sent to our teriaceae and (Nouioui et al. 2018). Cer- laboratory for bacteriological examination. tain members of the Corynebacterium are well The goat, the llama and the two alpacas lived all on known pathogens which spread within a herd and separate farms without any direct contact. cause abscesses (Braga et al. 2006, Sprake and Gold All samples were submitted for bacteriological exam- 2012), whereas bacterial species of the family Actinomy- ination to clarify the suspicion of CLA. Bacteriological cetaceae (actinomycetes) with the genera Actinomyces, examination was carried out according to standard pro- , Trueperella, and the recently described cedures. Abscess material was streaked on 5% sheep genus Schaalia (recently separated from the genus blood agar (Oxoid, Wesel, Germany) and MacConkey Actinomyces) usually affect individuals (Brown 2006, agar (BD BBL, Heidelberg, Germany) for aerobic incu- Fowler 1996, Nouioui et al. 2018). Therefore, detection bation at 37°C for two days. In addition, Schaedler agar of the causative agent and identification at species level (BD) and Wilkins-Chalgren agar with amikacin and 7% is of particular importance if there is suspicion of case- sheep blood (BD) were inoculated and incubated anaer- ous lymphadenitis (CLA) in goats, sheep, and camelids. obically at 37°C for two days. The isolates were stored in The family Actinomycetaceae forms a large group of our culture collection at -70°C using the Microbank™ anaerobic or microaerophilic bacteria producing short, system (Pro-Lab Diagnostics, Neston, Cheshire, U.K.). curved rods or branching filaments in varying degrees. For comparative studies, Schaalia hyovaginalis field Among these, the genera Actinomyces and Schaalia isolates and the type strain DSM 10695 (DSMZ German comprise many species which are widely distributed in Collection of Microorganisms and Cell Cultures, Braun- the environment and have been isolated from natural schweig, Germany) were included in this study (Table 1). habitats like , but also from humans and animals All bacterial isolates were analysed by matrix-assisted (Nouioui et al. 2018, Yassin 2014). Infections caused laser desorption ionization-time of flight mass spec- by actinomycetes have been recognised for some time trometry (MALDI-TOF MS) (Bruker Biotyper; Bruker (Smith 1918) and numerous species have since been Daltonik, Bremen, Germany), using the commercial described as causative agents of purulent and suppura- database 8.468 augmented with additional reference tive infections in various hosts (Nouioui et al. 2018, Yas- entries created in this study. The Bruker Biotyper data- sin 2014). However, due to the phylogenetic diversity of base 8.468 does not yet take into account the newer the genus Actinomyces which subdivides into different of the revised genus Actinomyces (Nouioui et clusters, lineages and groups, undescribed species are to al. 2018). be expected (Yassin 2014, Zhao et al. 2014). Accordingly, The creation of new reference entries, so called main the taxonomy of the Actinobacteria has undergone revi- spectra projections (MSP), followed the instructions and sion, species have been transferred to other genera and standards of the manufacturer. These procedures and the novel species have been proposed and defined in the software used have been described elsewhere in more last decade (Nouioui et al. 2018, Yassin 2014, Zhao et detail (Pranada et al. 2016, Rau et al. 2016a). Further al. 2014). In view of the large phylum of Actinobacteria information on user-made additional MSP applied in encompassing numerous pathogenic and apathogenic this study (see Table 1) is shown in the MALDI-UP cat- bacterial species, precise identification at species level is alogue on https://maldi-up.ua-bw.de (Rau et al. 2016b). crucial. This is of special relevance for the identification For decoding of 16S rRNA gene sequences, PCR of bacterial pathogens causing abscesses that resemble assays were carried out as described elsewhere (Contzen CLA when considering the impact on control programs et al. 2011) using the primers 27f (5’-AGA GTT TGA implemented for goats, sheep and camelids or assess- TCC TGG CTC AG-3’) and 1522rN (5’-CAT GCG GCC ment of successful treatment (Schumacher et al. 2009). GCA AGG AGG TGA TCC ARC CGC A-3’) according In this study, we describe the occurrence of abscesses to Johnson (1994). The PCR products were sequenced suspicious for CLA in a goat, a llama and two alpacas on demand (Microsynth, Balgach, Switzerland) and the caused by undescribed actinomycetes. These cases show sequence data obtained was compared with sequence that a broader spectrum of actinomycetes than previ- entries in GenBank (http://www.ncbi.nlm.nih.gov) using ously known poses a determining cause of abscess the Basic Local Alignment Search Tool for nucleotides formation and pyogenic lesions in small ruminants and (BLASTN) on the NCBI website (Pruitt et al. 2002). New World camelids. MALDI-TOF MS and 16S rDNA dendrograms were created using Actinomyces and Schaalia reference iso- lates including the isolates which originated from the Material and methods goat, llama and the two alpacas. In addition, a Coryne- bacterium pseudotuberculosis and a Trueperalla pyogenes In November 2011, material from an abscess on the neck strain were included (Table 1). of a llama (Lama glama) was submitted for bacteriologi- The MALDI-TOF MS dendrogram was created with cal examination. the “BioTyper MSP Dendrogram Creation Standard Berliner und Münchener Tierärztliche Wochenschrift 2020

TABLE 1: Data on the reference strains used in this study for cluster analysis of spectra obtained by MALDI-TOF MS and for construction of a phylogenetic tree based on 16S rRNA gene sequences Species Isolate (culture Source Acc. No. (culture collection no.) Sequence (bp) User- collection no.) made MSP* DSM 43014T cattle NR_118899 (DSM 43014T) 1379 yes Actinomyces bowdenii DSM 15435T dog NR_041982 (CCUG 37421T) 1513 Actinomyces dentalis DSM 19115T human NR_025633.1 (DSM 19115T) 1517 DSM 43320T human NR_114401 (JCM 12964T) 1512 DSM 43013T human NR_113326 (JCM 8349T) 1522

Actinomyces oris DSM 23056T human NR_117358 (ATCC 27044T) 1475

Actinomyces ruminicola DSM 27982T cattle NR_043523 (B71T) 1526 Actinomyces urogenitalis DSM 15434T human NR_25364 (CCUG 38702T) 1423 DSM 43327T hamster NR_026228 (ATCC 15987T) 1417

Actinomyces weissii DSM 24894T dog NR_108476 (CCM 7951T) 1379 yes

Corynebacterium pseudotuberculosis DSM 20689T sheep NR 119175.1 (NCTC 3450T) 1446 Schaalia canis DSM 15536T dog AJ243891 (CCUG 41706T) 1428 yes Schaalia cardiffensis DSM 15803T human NR_025521 (CCUG 44997T) 1522 yes Schaalia funkei DSM 15537T human NR_028960 (CCUG 42773T) 1366 Schaalia georgiae DSM 6843T human NR_026182 (DSM 6843T) 1422 Schaalia hyovaginalis CVUAS 30200 sheep MN864534 1494 yes Schaalia hyovaginalis CVUAS 4547.5 sheep MN864532 1494 yes Schaalia hyovaginalis CVUAS 6234.5 pig MN864533 1494 yes Schaalia hyovaginalis DSM 10695T pig MN864535 (DSM 10695T) 1497 yes Schaalia meyeri DSM 20733T human X82451 (CIP 103148T) 1441 Schaalia odontolytica DSM 19120T human NR_041983 (CCUG 20536T) 1412

Schaalia radingae DSM 9169T human NR_026169 (ATCC 51856T) 1429 Schaalia suimastitidis DSM 15538T pig NR_025401 (CCUG 39276T) 1416 Schaalia turicensis DSM 9168T human NR_037020 (ATCC 51857T) 1453 Schaalia vaccimaxillae DSM 15804T cattle NR_025523 (DSM 15804T) 1493 Trueperella pyogenes DSM 20630T pig NR 117537.1 (ATCC 19411T) 1293 yes

T = type strain; * own reference spectra (MSP) Further information is shown in the MALDI-UP catalogue on https://maldi-up.ua-bw.de.

Method”, as provided by the manufacturer within the terium (F.) necrophorum and Prevotella (P.) heparinolytica Biotyper software (vers. 3.1, Bruker), using the newly cre- detected under anaerobic atmospheric conditions. ated MSP from this study and a collection of MSP from The bacteria originating from the abscess of the llama the commercial Bruker MBT Compass Reference Library (isolate CVUAS 8688) and the alpacas (isolate CVUAS that was released in April 2019 and contains 2,969 spe- 31838 and CVUA 31845.2) showed profuse and strong cies and 8,468 MSP. growth of pinpoint-sized, non-haemolytic colonies on The phylogenetic tree was constructed using the blood sheep agar after a two-days incubation period neighbour-joining distance algorithm with standard set- in an aerobic atmosphere. None of the three isolates tings in Geneious Prime 2020.1.1. showed activity. Gram staining revealed gram- positive, short or slightly curved, club-shaped rods. Additionally, strong growth of Trueperella (T.) pyogenes Results and moderate growth of Bibersteinia (B.) trehalosi was observed with the llama’s abscess. In contrast, the isolates Bacterial culture originating from the alpacas grew in pure culture. The bacteria cultivated from the goat’s abscess (isolate CVUAS 31303) grew in a whitish, chalk-like layer of dis- MALDI-TOF MS analyses cernible pinpoint-sized, non-haemolytic colonies under The isolate CVUAS 31303 (goat) could be distinguished aerobic conditions. The bacteria appeared as catalase- from Schaalia isolates using MALDI-TOF MS and was positive, gram-positive, short, club-shaped rods. This located within the Actinomyces cluster (Fig. 1). The iso- primary culture was accompanied by an equivalent lates originating from the llama (isolate CVUAS 8688) strong growth of the obligate anaerobic bacteria Fusobac- and the alpacas (isolates CVUAS 31838 and CVUAS Berliner und Münchener Tierärztliche Wochenschrift 2020

31845.2) exhibit a close relationship clustering on a branch belonging to a separate cluster close to several Schaalia (S.) species, and are therefore referred to as Schaalia species. In contrast, caprine, sheep and porcine S. hyovaginalis field isolates and the S. hyovaginalis type strain DSM 10695, which were identi- fied and verified as S. hyovaginalis by MALDI-TOF MS, are located within a common cluster clearly separated from the isolates originating from the New World camelids (Fig. 1). The MALDI TOF mass-spectra of the isolates and several reference strains are available by exchange via the MALDI-TOF user platform (Rau et al. 2016b).

16S rRNA gene sequencing In the 16S rDNA dendrogam, the goat isolate CVUAS 31303 showed a close relationship to Actinomyces species, including Actinomyces bovis. In con- trast, the llama and alpaca isolates showed a close relationship in a com- mon sub-cluster within a Schaalia clus- ter, but were clearly distinct from the cluster comprising sheep, caprine and porcine S. hyovaginalis field isolates including the type strain DSM 10695 (Fig. 2). FIGURE 1: MALDI-TOF MS dendrogram created by cluster analysis of spectra obtained by MALDI-TOF mass spectrometry including the isolates Discussion CVUAS 31303 (goat), CVUAS 8688 (llama), CVUAS 31845.2 (alpaca) and CVUAS 31838 (alpaca) in context of reference spectra. For details of the isolates A considerable proportion of abscess and reference spectra used in this study see Table 1. formation in small ruminants and camelids is caused by bacteria belong- For this dendrogram, the same isolates were used as for the MALDI-TOF mass ing to the phylum Actinobacteria. A spectrometry dendrogram (Fig. 1). For details of the isolates and sequences used prominent member of the order Actin- in this study see Table 1 (Graphic: CVUA Stuttgart). omycetales is Corynebacterium (C.) pseudotuberculosis, the causative agent of CLA, which is highly relevant in small ruminants 2018, Fielding et al. 2008), dogs (Hoyles et al. 2000, Song and camelids worldwide (Al-Harbi 2011, Al-Tuffyli and et al. 2015) and captive and free-ranging wildlife (Alssa- Shekhan 2012, de la Fuente et al. 2017, de Lima e Silva et hen et al. 2020, Gamble and Clancy 2013, Wickhorst et al. al. 2016). Control programmes have been implemented 2017). The microorganisms reported in this study, which in numerous countries worldwide to effectively combat had been isolated from abscesses in a goat, a llama and CLA in small ruminants and camelids. Therefore, abscess two alpacas, attracted our attention because of their formation resembling CLA has to be verified by identi- similarity to Actinomyces spp. and Schaalia spp., respec- fication of the pathogenic agent, which must be reliably tively. Further studies on these isolates using MALDI- distinguished from C. pseudotuberculosis. Numereous TOF MS and 16S rDNA analysis initially yielded incon- actinomycetes have been described as causative agents of clusive results at species level, despite comprehensive suppurative lesions in humans and animals (Nouioui et databases that are available for evaluation procedures. al. 2018, Yassin 2014). Among the genera within the fam- However, the isolates could be assigned to bacteria of ily Actinomycetaceae, the recently described novel genus the genera Actinomyces (goat isolate) and Schaalia (llama Schaalia has been separated from the genus Actinomyces and alpaca isolates). The isolate originating from the and reported in connection with abscess formation in abscess in the goat is located within the MALDI-TOF humans and various animal species. Case reports have MS and 16S rDNA Actinomyces cluster, but is separate to been published on abscess formation due to Actinomyces other concrete Actinomyces spp. In contrast, the isolates and Schaalia infections in different localisations such as obtained from the llama and the alpacas show very close the brain, subcutis or lymph nodes of goats (Alssahen et relationships to each other in a separate branch next to al. 2020, Hirai et al. 2007, Ndegwa et al. 2001, Oyekunle other Schaalia spp. in both the MALDI-TOF MS and 16S et al. 2010, Schumacher et al. 2009), New World camelids rDNA dendrogram. The close relationship of these actin- (Brown 2006, Fowler 1996), sheep (Alssahen et al. 2020, omycetes in New World camelids, which were isolated Collins et al. 2001, Foster et al. 2012), pigs (Hommez et at different times and places, suggest the presence of a al. 1991, Reichel and Wragg 2007), horses (Chung et al. novel host-adapted Schaalia species. Fowler (1996) also Berliner und Münchener Tierärztliche Wochenschrift 2020

FIGURE 2: Phylogenetic tree based on 16S rRNA gene sequences including the isolates CVUAS 31303 (goat, 1487 bp; GenBank Accession No. MN864531), CVUAS 8688 (llama, 1517 bp; MN864528), CVUAS 31845.2 (alpaca, 1517 bp; MN864529) and CVUAS 31838 (alpaca, 1517 bp; MN864530). Numbers indicate percent bootstrap sup- port (100 replications). For this dendrogram, the same isolates were used as for the MALDI-TOF mass spectrometry dendrogram (Fig. 1). For details of the isolates and sequences used in this study see Table 1 (Graphic: CVUA Stuttgart). reported on abscess formation in the throat region, lungs Conclusion or liver and dental abscesses caused by Actinomyces sp. unique to New World camelids and provisionally called Actinomycetes represent important bacteria associ- these isolates Actinomyces (lamae). The author pointed ated with abscess formation. This report shows that out that these bacterial isolates may be under-reported so far undescribed actinomycetes are also responsible due to their morphological appearance as gram-positive, for abscess formation in addition to already described short rods which may be mistaken for cocci. pyogenic bacteria. Precise and reliable identification at It is notable that the actinomycetes growth reported species level is therefore crucial with special reference to in this study was accompanied by F. necrophorum and CLA (pseudotuberculosis), which is controlled in large- P. heparinolytica (goat) or T. pyogenes and B. trehalosi scale control programmess in many countries. Reliable (llama). However, no accompanying bacterial aerobic or identification of Actinomyces and Schaalia species and anaerobic flora could be detected in the abscesses from detection of undescribed species can be achieved by the two alpacas. Other researchers also report growth of MALDI-TOF MS and 16S rDNA analyses. actinomycetes in pure cultures (Brown 2006, Chung et al. The present study extends our current knowledge 2018, Foster et al. 2012, Oyekunle et al. 2010, Song et al. about novel actinomycetes associated with abscess for- 2015) or growth in mixed cultures including T. pyogenes, mation in small ruminants and New World camelids. Fusobacterium spp., Bacteroides spp. and Prevotella spp. or Staphylococcus spp. and Streptococcus spp. (Al-Harbi 2011, Collins et al. 2001, Foster et al. 2012, de la Fuente Acknowledgement et al. 2017, Gamble and Clancy 2013, Roeder et al. 1989, Schumacher et al. 2009, Wickhorst et al. 2017). The authors thank Jana Ade and Mandy Hailer for The abscess wounds in the two alpacas were successfully performance and evaluation of the PCR assays. We treated by rinsing with iodine solution without additional would also like to thank Martin Dyk for carrying out antibiotic treatment. Unfortunately, no data is available on the MALDI-TOF MS analyses and the evaluation of the the treatment of the abscesses in the goat and the llama. spectra. MALDI-TOF MS and 16S rRNA gene analyses sug- gest that the actinomycetes described in this study represent undesignated novel Actinomyces and Schaalia Conflict of interest statement species. However, this assumption has to be proven by further comprehensive investigations to elucidate the None of the authors has any financial or personal rela- taxonomic status of these interesting, obviously patho- tionships that could inappropriately influence or bias the genic actinomycetes. content of this paper. Berliner und Münchener Tierärztliche Wochenschrift 2020

References Ndegwa EN, Muleib CM, Munyua SJM (2001): Prevalence of microorganisms associated with udder infections in dairy goats Al-Harbi KB (2011): Prevalence and etiology of abscess disease on small-scale farms in Kenya. J S Afr Vet Assoc 72: 97–98. of sheep and goats at Qassim region, Saudi Arabia. Veterinary Nouioui I, Carro L, Garcia-Lopez M, Meier-Kolthoff JP, Woyke World 4, 495–499. T, Kyrpides NC, Pukall R, Klenk HP, Goodfellow M, Goker Al-Tuffyli YIK, Shekhan MI (2012): Clinical and bacteriological M (2018): Genome-based taxonomic classification of the phy- study of subcutaneous abscesses caused by gram positive bac- lum Actinobacteria. Front Microbiol 9: 2007. teria in cow and sheep in Al-Qadissiyia province. AL-Qadisiyah Oyekunle MA, Talabi AO, Agbaje M, Oni OO, Adebayo AO, Journal of Vet Med Sci 11: 80–85. Olude MA, Oyewusi IK, Akinduti PA (2010): in West African dwarf goat in Nigeria. Niger Vet J 31: 80–86. Alssahen M, Hassan AA, Rau J, Sammra O, Wickhorst J-P, Lämmler C, Prenger-Berninghoff E, Eisenberg T, Abdul- Pranada BP, Schwarz G, Kostrzewa M (2016): MALDI Biotyping mawjood A (2020): Comparative studies on Schaalia (Actino- for microorganism identification in clinical microbiology. In: myces) hyovaginalis isolated from wild boar, goat and sheep. Berl Cramer R (ed) Advances in MALDI and Laser-induced soft Münch Tierärztl Wochenschr Online first: 31.03.2020. http:// ionization mass spectrometry, 1st ed. Springer International vetline.de/facharchiv/158/3222 Publishing, Basel. Braga WU, Chavera A, Gonzalez A (2006): Corynebacterium pseu- Pruitt K, Brown G, Tatusova T, Maglott D (2002): The Reference dotuberculosis infection in highland alpacas (Lama pacos) in Sequence (RefSeq) Database (Updated 2012 Apr 6) In: McEn- Peru. Vet Rec 159: 23–24. tyre J, Ostell J (eds.). The NCBI Handbook [Internet]. Bethesda (MD). National Center for Biotechnology Information (US), Brown RA (2006): Unusual findings in a llama. Vet Rec 159: Chapter 18. 755–756. Rau J, Eisenberg T, Männig A, Wind C, Lasch P, Sting R (2016a): Chung ELT, Adamu L, Jesse FFA, Wakil YK, Solomon EM, MALDI-TOF mass spectrometry for reliable identification of Turaki UA (2018): Suspected neonatal isoerythrolysis with bacteria – A validation based on Staphylococcaceae field isolates. concurrent Actinomyces hyovaginalis in a foal. J Adv Vet Anim Aspects of Food Control and Animal Health (eJournal)2016-03: Res 5: 233-239. 1–46. https://ejournal.cvuas.de/issue201603.asp Collins MD, Hutson RA, Hoyles L, Falsen E, Nikolaitchouk N, Rau J, Eisenberg T, Sting R (2016b): MALDI-UP – an internet Foster G (2001): Streptococcus ovis sp. nov., isolated from sheep. platform for the exchange of MALDI-TOF mass spectra (eJour- Int J Syst Evol Microbiol 51: 1147–1150. nal). 2016-01: 1–17. https://ejournal.cvuas.de/issue201601.asp Contzen M, Sting R, Blazey B, Rau J (2011): Corynebacterium Reichel R, Wragg P (2007): of Actinomyces hyovaginalis ulcerans from diseased wild boars carrying Corynebacterium from a lung lesion in a pig. Vet Rec 160: 203. diphteriae-like tox genes. Zoonoses Public Health 58: 479–488. Roeder BL, Chengappa MM, Lechtenberg KF, Nagaraja TG, Fielding CL, Magdesian KG, Morgan RA, Ruby RE, Sprayberry Varga GA (1989): Fusobacterium necrophorum and Actinomyces KA (2008): Actinomyces species as a cause of abscesses in nine pyogenes associated facial and mandibular abscesses in blue horses. Vet Rec 162: 18–20. duiker. J Wildl Dis 25: 370–377. Foster G, Wragg P, Koylass MS, Whatmore AM, Hoyles L Schumacher VL, Hinckley L, Gilbert K, Risatti GR, Londoño (2012): Isolation of Actinomyces hyovaginalis from sheep and AS, Smyth JA (2009): Actinomyces hyovaginalis-associated comparison with isolates obtained from pigs. Vet Microbiol 157: lymphadenitis in a Nubian goat. J Vet Diagn Invest 21: 380–384. 471–475. Smith T (1918): A pleomorphic from pneumotic lungs of Fowler ME (1996): Husbandry and diseases of camelids. Scientific calves simulating Actinomyces. J Exp Med 28: 333–344. and Technical Review of the International Office of Epizootics Song, RB, Vitullo, CA, da Costa, RC, Daniels, JB (2015): Long- 15: 55–169. term survival in a dog with meningoencephalitis and epidural de la Fuente R, de las Herasa M, Torrijosa C, Diez de Tejadaa abscessation due to Actinomyces species. J Vet Diagn Invest 27: P, Pérez-Sanchob M, Carrióna FJ, Ordena JA, Dominguez- 552–557. Bernala G (2017): Short communication: Isolation frequency Sprake P, Gold JR (2012): Corynebacterium pseudotuberculosis liver of bacteria causing lymphadenitis and abscesses in small rumi- abscess in a mature alpaca (Lama pacos). Can Vet J 53: 387–390. nants in central Spain. Small Ruminant Res 154: 5–8. Wickhorst J, Sammra O, Hassan A, Alssahen M, Lämmler C, de Lima e Silva WE, Veneroni Gouveia G, da Conceição Aquino Riße K, Eisenberg T, Schauerte N, Geiger C, Prenger-Bern- de Sá M., de Simoni Gouveia JJ, de MoraesPeixoto R, Riet- inghoff E, Timke M, Abdulmawjood A (2017): Actinomyces Correa F, Veschi JLA, da Costa MM (2016): Bacteria isolated hyovaginalis associated with chronic suppurative arthritis in from abscesses of small ruminants inspected in the semiarid an adult giraffe (Giraffa cameloperdalis reticulate). Berl Münch region of Brazil. J Semina Ciênc Agrár 37: 1337–1344. Tierärztl Wochenschr 130: 161–164. Gamble KC, Clancy MM (2013): Infectious diseases of concern Yassin AAF (2014): The Family Actinomycetaceae. In: The Prokary- to captive and free ranging animals in North America. 2nd ed. otes, 4th ed. Springer, Berlin Heidelberg. Infectious Disease Committee, American Association of Zoo Zhao K, Li W, Kang C, Du L, Huang T, Zhang X, Wu M, Yue B Veterinarians, Yulee, Florida (2014): Phylogenomics and evolutionary dynamics of the family Hirai T, Nunoya T, Azuma R (2007): Actinomycosis of the brain Actinomycetaceae. Genome Biol Evol 6: 2625–2633. and temporal bone in a goat. J Vet Med Sci 69: 641–643. Hoyles L, Falsen E, Foster G, Pascual C, Greko C, Collins MD (2000): Actinomyces canis sp. nov., isolated from dogs. Int J Syst Address for correspondence Evol 50 Pt 4: 1547–1551. Dr. Reinhard Sting Hommez J, Devriese LA, Miry C, Castryck F (1991): Charac- Chemisches und Veterinäruntersuchungsamt Stuttgart terization of 2 groups of Actinomyces-like bacteria isolated from Schaflandstr. 3/3 purulent lesions in pigs. Zentralbl Veterinarmed B 38: 575–580. 70736 Fellbach Johnson JL (1994): Similarity analysis of rRNAs. In: Methods for Germany General and Molecular Bacteriology, 2nd ed. American Society [email protected] for Microbiology, Washington DC.